Expression and Characterization of α-Methylacyl CoA Racemase from Anisakis simplex Larvae

Larval excretory-secretory products of Anisakis simplex are known to cause allergic reactions in humans. A cDNA library of A. simplex 3rd-stage larvae (L3) was immunoscreened with polyclonal rabbit serum raised against A. simplex L3 excretory-secretory products to identify an antigen that elicits the immune response. One cDNA clone, designated as α-methylacyl CoA racemase (Amacr) contained a 1,412 bp cDNA transcript with a single open reading frame that encoded 418 amino acids. A. simplex Amacr showed a high degree of homology compared to Amacr orthologs from other species. Amacr mRNA was highly and constitutively expressed regardless of temperature (10-40℃) and time (24-48 hr). Immunohistochemical analysis revealed that Amacr was expressed mainly in the ventriculus of A. simplex larvae. The Amacr protein produced in large quantities from the ventriculus is probably responsible for many functions in the development and growth of A. simplex larvae

Blood-Stage Plasmodium Berghei ANKA Infection Promotes Hepatic Fibrosis by Enhancing Hedgehog Signaling in Mice

Background/Aims: Malaria is the most deadly parasitic infection in the world, resulting in damage to various organs, including the liver, of the infected organism; however, the mechanism causing this damage in the liver remains unclear. Liver fibrosis, a major characteristic of liver diseases, occurs in response to liver injury and is regulated by a complex network of signaling pathways. Hedgehog (Hh) signaling orchestrates a number of hepatic responses including hepatic fibrogenesis. Therefore, we investigated whether Hh signaling influenced the liver’s response to malarial infection. Methods: Eight-week-old male C57BL/6 mice inoculated with blood containing Plasmodium berghei ANKA (PbA)-infected erythrocytes were sacrificed when the level of parasitemia in the blood reached 10% or 30%, and the livers were collected for biochemical analysis. Liver responses to PbA infection were examined by hematoxylin and eosin staining, real-time polymerase chain reaction, immunohistochemistry and western blot. Results: Severe hepatic injury, such as ballooned hepatocytes, sinusoidal dilatation, and infiltrated leukocytes, was evident in the livers of the malaria-infected mice. Hypoxia was also induced in 30% parasitemia group. With the accumulation of Kupffer cells, inflammation markers, TNF-α, interleukin-1β, and chemokine (C-X-C motif) ligand 1, were significantly upregulated in the infected group compared with the control group. Expression of fibrotic markers, including transforming growth factor-β, α-smooth muscle actin (α-SMA), collagen 1a1, thymosin β4, and vimentin, were significantly higher in the infected groups than in the control group. With increased collagen deposition, hepatic stellate cells expressing α-SMA accumulated in the liver of the PbA-infected mice, whereas those cells were rarely detected in the livers of the control mice. The levels of Hh signaling and Yes-associated protein (YAP), two key regulators for hepatic fibrogenesis, were significantly elevated in the infected groups compared with the control group. Treatment of mice with Hh inhibitor, GDC-0449, reduced hepatic inflammation and fibrogenesis with Hh suppression in PbA-infected mice. Conclusion: Our results demonstrate that HSCs are activated in and Hh and YAP signaling are associated with this process, contributing to increased hepatic fibrosis in malaria-infected livers

Seroprevalence of Antibodies against Anisakis simplex Larvae among Health-Examined Residents in Three Hospitals of Southern Parts of Korea

The present study was performed to estimate the seroprevalence of larval Anisakis simplex infection among the residents health-examined in 3 hospitals in southern parts of Korea. A total of 498 serum samples (1 serum per person) were collected in 3 hospitals in Busan Metropolitan city, Masan city, and Geoje city in Gyeongsangnam-do (Province) and were examined by IgE-ELISA and IgE-western blotting with larval A. simplex crude extract and excretory-secretory products (ESP). The prevalence of antibody positivity was 5.0% and 6.6% with ELISA against crude extracts and ESP, respectively. It was also revealed that infection occurred throughout all age groups and higher in females than in males. A specific protein band of 130 kDa was detected from 10 patients with western blot analysis against crude extract and ESP among those who showed positive results by ELISA. Our study showed for the first time the seroprevalence of anisakiasis in Korea. The allergen of 130 kDa can be a candidate for serologic diagnosis of anisakiasis

Hepatic stellate cells express thymosin Beta 4 in chronically damaged liver.

Although the various biological roles of thymosin β4 (Tβ4) have been studied widely, the effect of Tβ4 and Tβ4-expressing cells in the liver remains unclear. Therefore, we investigated the expression and function of Tβ4 in chronically damaged livers. CCl4 was injected into male mice to induce a model of chronic liver disease. Mice were sacrificed at 6 and 10 weeks after CCl4 treatment, and the livers were collected for biochemical analysis. The activated LX-2, human hepatic stellate cell (HSC) line, were transfected with Tβ4-specific siRNA and activation markers of HSCs were examined. Compared to HepG2, higher expression of Tβ4 in RNA and protein levels was detected in the activated LX-2. In addition, Tβ4 was up-regulated in human liver with advanced liver fibrosis. The expression of Tβ4 increased during mouse HSC activation. Tβ4 was also up-regulated and Tβ4-positive cells were co-localized with α-smooth muscle actin (α-SMA) in the livers of CCl4-treated mice, whereas such cells were rarely detected in the livers of corn-oil treated mice. The suppression of Tβ4 in LX-2 cells by siRNA induced the down-regulation of HSC activation-related genes, tgf-β, α-sma, collagen, and vimentin, and up-regulation of HSC inactivation markers, ppar-γ and gfap. Immunofluorescent staining detected rare co-expressing cells with Tβ4 and α-SMA in Tβ4 siRNA-transfected cells. In addition, cytoplasmic lipid droplets were observed in Tβ4 siRNA-treated cells. These results demonstrate that activated HSCs expressed Tβ4 in chronically damaged livers, and this endogenous expression of Tβ4 influenced HSC activation, indicating that Tβ4 might contribute to liver fibrosis by regulating HSC activation

Detection of Tβ4 in the activated HSCs and human fibrotic liver.

<p>(A) QRT-PCR analysis of mRNA form human hepatic stellate cell line (LX-2) and human hepatocellular carcinoma cell line (HepG2). Mean±SD results are graphed. Data represent the mean±SD of three independent experiments (**p<0.005 vs. HepG2). (B) Western blot analysis of Tβ4 (GAPDH was used as an internal control). Data shown represent one of three experiments with similar results. (C) Representative immunostaining images for Tβ4 in LX-2 cells and HepG2 cells. Brown color indicated Tβ4-positive cells. (X40) (D) QRT-PCR analysis of mRNA from mouse primary HSCs (Day 0: freshly isolated HSCs / Day 7: cultured primary HSCs for 7 days. Mean±SD results are graphed. Data represent the mean±SD of three independent experiments (**p<0.005). (E) QRT-PCR analysis of RNA isolated from healthy livers (HL, n = 3) and fibrotic livers (stage 4 of fibrosis, n = 13). Mean±SD results are graphed and data represent the mean±SD of three independent experiments (**p<0.005).</p

siRNA for Tβ4 blocks activation of LX-2 cells.

<p>(A) RNA and protein expression of Tβ4 in control siRNA-, Tβ4 siRNA- or non-treated LX-2 cells. Data represent the mean±SD of three independent experiments. GAPDH was used as an internal control (*p<0.05; **p<0.005). (B) QRT-PCR analysis for tgf-β, α-sma, col1α1, vimentin, ppar-γ and gfap. Mean±SD results are graphed. Data represent the mean±SD of three independent experiments (*p<0.05; **p<0.005 vs. control siRNA). (C) Double immunofluorescent staining for Tβ4 and α-SMA in Tβ4 siRNA-treated and control siRNA-treated cells (X20) and Oil Red O staining in siRNA-treated and control siRNA-treated cells (X40). ORO-positive cells were expressed as percentage of ORO-positive cells/total cells. Data represent the mean±SD of three independent experiments (**p<0.005). (Tβ4 siRNA: Tβ4 siRNA-treated LX-2 cells, Control siRNA: Control siRNA-treated LX-2 cells)</p

Up-regulation of Tβ4 in the livers from CCl₄-treated mice.

<p>(A) QRT-PCR analysis of liver mRNA from CCl₄-treated mice for Tβ4 (n = 4 mice / group). Mean±SD results are graphed. (B) and (C) Western blot analysis of Tβ4 (GAPDH was used as an internal control). Data shown represent one of three experiments with similar results. (B: Immunoblot / C: Band density) (n≥3 mice/ group) Data represent the mean±SD of three independent experiments (**p<0.005 vs. own control group). (D) POP activities in homogenates of controls and CCl4-treated mice are presented in picomole of AMC/min x mg tissue. Mean±SD results are graphed and data represent the mean±SD of three independent experiments (**p<0.005 vs. own control group). (E) IHC for Tβ4 in the liver at 6 and 10 weeks post CCl₄ injection. Brown color indicated Tβ4-positive cells (X40). Magnified representative images from CCl₄-treated livers at 6 and 10 weeks (X63) is shown in right panel. TB4-positive cells were quantified by dividing of the total number of positive cells by the total numbers of hepatocytes per field. Mean±SD results are graphed (**p<0.005 vs. own control group). (CON: corn oil-treated mice, CCl₄: CCl₄-treated mice).</p